PEGylation of drug candidate proteins or nucleic acids have been useful for improving biokinetics thereof (in vivo retention, stability against proteases or nucleases, immunogenicity, etc). Numerous PEGylated proteins have been approved by FDA as medicines and many have been under development as well. Approved and commercialized medicines typically include PEG-asparaginase, PEG-adenosine deaminase, PEG-interferon α-2a, PEG-interferon α-2b, PEG-G-CSF, PEG-growth hormone receptor antagonists and nonproteinaceous medicines such as branched PEG-anti-VEGF aptamers. Among others, branched PEGs which become a branched form after binding to proteins have been recently attracting attention and contributing to development of new PEGylated medicines under recent difficult conditions to develop novel medicines (Non-Patent Document 1).
Most of branched PEGs used for PEGylated medicines so far are functionalized with active esters through which the PEGs can react with an amino group in proteins. However, the active esters generally react not only with lysine in proteins but also with histidine. Further during PEGylation in which PEGs having active ester terminal groups react with amino groups of proteins, the formation of an amide bond may significantly change the basicity (or the extent of positive charge) of the amino groups, resulting in significant decrease in protein activity. Thus there has been a need for establishment of the production technique of PEGylated proteins while maintaining the amino group basicity (or the extent of positive charge). The synthesis and purification of conventional branched PEGs is also disadvantageous in that it includes complex steps. Because of these problems of harsh reaction conditions, low reaction efficiency and generation of byproducts associated with the conventional PEGylation techniques, many proteins have not reached successful development in spite of expectation therefor as efficacious medicines.